Hello all,
i wanted to know whether its possible to culture a particular mutant cell line which over expresses a protein X (proved), lyse them and treat the lysate with varying concentrations of drugs and immunoblot later on for the presence of protein X???
Thanks!!!
It is certainly possible to do what you propose, but what is the purpose of the experiment? Once the cells are lysed, there will be no further protein synthesis. There may be degradation, but it could have multiple causes.
there is no point in treating the lysate with any chemical agent as when the cell lysis occurs, all the pathways are inhibited.
You can make an attempt to do pull down assay. I have a protocol for you. Hope this will be helpful:
http://www.nature.com/nmeth/journal/v1/n3/full/nmeth1204-275.html
Without having a clear picture of your goal, it is difficult to know what to suggest. However, it seems to me that you should treat the cells with the drugs before lysis, probably for several hours to a day, if you are expecting a change in protein level. On the other hand, if you are looking for a change in a reversible protein-protein interaction, then it might work just as well to use a cell lysate.
Hi I am planning on doing a similar experiment where I want to treat cell lysate with drugs to look for protein interactions. What would be the average incubation time with the drug on the cell lysate?
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